Skeletal muscle activity is regulated by electrical signals propagated by motor neurons which innervate muscle at the neuromuscular junction (NMJ), a specialized synapse structure (1). Nicotinic acetylcholine receptors (nAChRs) positioned on the post-synaptic membrane of NMJ are central in mediating nerve-muscle communication (2). As such, nAChRs are regulated at both transcriptional and post-translational levels in response to neural activity. Neural input affects NMJ stability and function through multiple mechanisms (1). First, the nerve secreted protein agrin induces nAChR clustering through activation of a MUSK/rapsyn/nAChR complex, which engages the cytoskeleton and provides an extremely high local density of nAChR at the NMJ (3, 4). Members of the Dystrophin-Glycoprotein complex (DGC) are critical in providing stability and maintenance of the synapse (5, 6). Second, nAChR gene expression is induced specifically in sub-synaptic nuclei located beneath the post-synaptic apparatus by the transcription factor GABP, a process which acts to locally deposit activity-dependent factors at the NMJ, including nAChR, MUSK, and structural proteins (7, 8). Finally, to exclude the expression of nAChR subunits in extra-synaptic regions, activity-mediated calcium efflux activates calcium-dependent enzymes, such as calcium-dependent protein kinase II (CaMKII) (9-12), which through a yet to be characterized pathway, down-regulates myogenin expression and therefore inhibits nAChR expression as a consequence. In conjunction, these three processes account for much of the synaptic accumulation of nAChRs.
Conversely, reduced neural activity by surgical denervation, inactivity or neuromuscular defects associated with pathological conditions, such as Amyotrophic Lateral Sclerosis (ALS), can lead to extrasynaptic expression of nAChR induced by the bHLH transcription factor myogenin (MGN) (13). Such activation of transcription similarly occurs during embryonic muscle development before innervation, leading to high level expression of nAChR subunits throughout the muscle. The increased levels of AChR result in acetylcholine super-sensitivity, which is thought to be important for successful formation of muscle synapse after innervation. This induction of nAChR is mediated by myogenin, which binds E-box elements (CANNTG) present in the promoters of activity-regulated genes including nAChRε, nAChR and MUSK (14-19). Myogenin gene expression is repressed by Dachschund related transcriptional co-repressor, Dach2 (20). Interestingly, Dach2 expression itself is suppressed by denervation, suggesting a model whereby reduction of Dach2 causes transcriptional induction of myogenin and subsequent activation of nAChR (20).
Myofibers are grossly classified into slow/oxidative fibers and fast/glycolytic fibers that are distinct in contractile velocity and metabolic properties. The slow/oxidative fibers are slow in twitching but more resistant to fatigue. Metabolically, they are rich in mitochondria, and use oxidative phosphorylation to provide ATP for contraction (thus termed oxidative fibers). The fast/glycolytic fibers, in contrast, are fast in contractility but prone to fatigue. They contain fewer mitochondria and use glycolysis as the main source of energy (Spangenburg E E, Booth F W. Acta Physiol Scand. (2003) 178(4):413-24). In addition to contractile properties, these fibers also differ in their capacity in glucose metabolism with slow/oxidative fibers being more responsive to insulin than fast/glycolytic fibers. Thus, the composition of myofibers is not only critical for performing different movements but also important for regulating glucose metabolism.
The functionality of a myofiber is determined by the expression of unique sets of contractile proteins and metabolic enzymes at the transcriptional level. For example, in slow fibers, type I MHC is the dominant form of myosin heavy chain while in fast fiber, type IIb or IIx MHC are the main isotypes (Spangenburg E E, Booth F W. Acta Physiol Scand. (2003) 178(4):413-24). On the metabolic end, the differential expression of the transcriptional co-activator PGC-1α, which promotes mitochondrion biogenesis, likely plays a critical role (Lin J et al., Cell Metab. (2005) 1(6):361-70). Thus, PGC-1α is expressed at a higher level in mitochondrion-rich slow/oxidative fibers than in fast/glycolytic fibers. In fact, over-expression of PGC-1α is sufficient to convert fast/glycolytic fibers to slow/oxidative fibers in a mouse model (Lin J. et al., Nature. (2002) 418(6899):797-801).
Uniquely, the adult myofiber phenotype is not permanent. The size, contractility and metabolic property of myofibers can all undergo changes or remodeling (Talmadge R J. Muscle Nerve. (2000) 23(5):661-79). For example, reduced neuromuscular activity caused by inactivity, ageing or neuromuscular disease can lead to reduction in muscle size, termed atrophy. Interestingly, inactivity also causes an increase in fast/glycolytic fibers. Conversely, repetitive use, such as exercise training, induces muscle hypertrophy and a concomitant increase in oxidative/slow fiber. These coordinated changes allow muscle to perform different tasks and meet different functional demands. This remarkable adaptability of skeletal muscle is achieved by a highly regulated gene transcription program, which in response to differential neuromuscular activities, controls myofiber size, contractility and metabolism. This so called “neural activity-dependent muscle remodeling” is essential for muscle function and homeostasis.
Temporary reduction in muscle size is part of normal remodeling in responses to reduced neural activity. However, chronically reduced neural activities associated with neuromuscular disease, aging and denervation can cause muscle atrophy, a muscle wasting disease characterized by an excessive reduction in muscle size and strength. In neuromuscular diseases, such as Amyotrophic Lateral Sclerosis (ALS), motor neuron dysfunction leads to severe muscle atrophy in both diaphragm and limbs, contributing to breathing and moving difficulty and eventual death. The loss of muscle mass in atrophy is, at least in part, due to accelerated protein degradation catalyzed by two ubiquitin E3 ligases, atrogin-1/MAFbx and MuRF1, both of which are induced transcriptionally in atrophic muscle (Bodine S C, et al., Science. (2001) 294(5547):1704-8); Gomes M D, et al, Proc Natl Acad Sci USA. (2001) 98(25): 14440-5). Although the detailed mechanism remains uncertain, Atrogin-1 and MURF1 were proposed to promote atrophy by degrading structural and contractile proteins, such as myosin light chain, myotilin, myomesin and titin (Witt S H, et al., J Mol Biol. 2005; 350(4):713-22). Interestingly, gene array analysis revealed that the same set of genes is also transcriptionally repressed in atrophic muscle (Bodine S C, et al., Science. (2001) 294(5547):1704-8). These observations indicate that the atrophy-associated decrease in structural and contractile proteins might be achieved through both active protein degradation and transcriptional repression caused by reduced neural activity. Identifying the transcriptional pathway responsible for atrophy-associated gene repression could provide new therapeutic opportunity for this devastating disease.
Pathological muscle remodeling has also been implicated in the development of metabolic disease. A decrease in oxidative/slow and an increase in glycolytic/fast fibers have been reported in patients with type II diabetes (Oberbach A, et al., Diabetes Care. (2006) 29(4):895-900). Indeed, aging and inactivity, which are both associated with the development of insulin resistance, also lead to diminished oxidative capacity of skeletal muscle. Conversely, an increase in slow/oxidative fibers by exercise training enhances insulin sensitivity (Oberbach A, et al., Diabetes Care. (2006) 29(4):895-900). Thus, the composition of myofibers is important for regulating glucose metabolism. A pharmacological means of stimulating oxidative fiber formation in muscle—an ability to control fiber type specification by manipulating the muscle-remodeling program—could have the potential to increase insulin sensitivity and thereby provide therapeutic benefits to patients with type II diabetes. Elucidating the transcriptional network that coordinately controls myofiber type and size would be a key toward the development of and effective therapeutic strategy.
Six histone deacetylases have been identified in mammalian cells, the yeast RPD3 homologs HDAC1, HDAC2 and HDAC3 and the yeast HDA1 homologs HDAC4/HDAC-A, HDAC5 and HDAC6.
Histone deacetylase 4 (also known as HDAC4 and HDAC-A) was the first HDA1 homolog to be identified (Fischle et al., J. Biol. Chem., 274:11713-11720 (1999); Grozinger et al., Proc. Natl. Acad. Sci. USA, 96:4868-4873 (1999); Miska et al., Embo J., 18:5099-5107 (1999)). Unlike the other histone deacetylases, histone deacetylase 4 contains a noncatalytic N-terminal domain through which it can interact with specific transcription factors. When tethered to a promoter, histone deacetylase 4 has been shown to represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain found in the C-terminus (Wang et al., Mol. Cell. Biol., 19:7816-7827 (1999)).
Another unique feature described for histone deacetylase 4, involves its cellular localization. The protein shuttles between the nucleus and cytoplasm in a process involving active transport. In the nucleus, through a small region located at its N-terminal domain, histone deacetylase 4 interacts with two MADS-box transcription factors, MEF2A (Miska et al., Embo J., 18:5099-5107 (1999)) and MEF2C (Wang et al., Mol. Cell. Biol., 19:7816-7827 (1999)). Furthermore, histone deacetylase 4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results indicate that HDAC4 interacts with transcription factors such as MEF2C and MEF2A to negatively regulate gene expression. Co-immunoprecipitation data demonstrated that histone deacetylase 4 does not interact with either the NURD or mSin3A complexes as do other histone deacetylases (Grozinger et al., Proc. Natl. Acad. Sci. USA, 96:4868-4873 (1999)). However, it has been found in association with other proteins (Fischle et al., J. Biol. Chem., 274:11713-11720 (1999)).
Huang et al. have shown that histone deacetylase 4 represses transcription by interacting with the conserved nonredundant repression domains (RDs) found in the corepressors, N-CoR and SMRT. These corepressors play a critical in the function of nuclear hormone receptors, and are deregulated in human myeloid leukemias. Endogenous N-CoR and SMRT each associate with HDAC4 in a complex that does not contain mSin3A or HDAC1. These results indicate that alternative histone deacetylase complexes may mediate specific repression pathways in normal as well as leukemic cells (Huang et al., Genes Dev., 14:45-54 (2000)).
Histone deacetylase 4 (HDAC4) is expressed in various adult human tissues, with the highest levels of expression found in skeletal muscle, thymus and small intestine and very low levels found in liver, placenta and kidney (Fischle et al., J. Biol. Chem., 1999, 274, 11713-11720).